A major area of research in regenerative medicine is the potential application of cell therapy for various disorders. Embryonic stem (ES) cells are self-renewable and pluripotent cells derived from the inner cell mass of a blastocyst-stage embryo. ES cells have the potential to provide innovative therapeutic options for a wide variety of degenerative and non-degenerative human disorders, including the replacement of dysfunctional airway epithelia. To achieve this goal, it would require the development of well defined and efficient protocols for directing the commitment and differentiation of stem cells into the airway epithelial cell lineage, together with their selective purification and proliferation in vitro. The development of such protocols would reduce the likelihood of spontaneous differentiation of ES cells into divergent lineages upon transplantation, as well as reduce the risk of teratoma formation. Additional, such protocols could provide useful in vitro models for studying the developmental biology, of respiratory tract epithelial cells, as well as facilitate the genetic manipulation of stem cells for therapeutic application. The hypothesis of this application is that ES cells can be in vitro trained to differentiate to the airway epithelial cell lineages that have the full potential to regenerate airway epithelium in vivo. To test this hypothesis, three approaches are undertaken. 1) Define "minimally optimized serum-free culture conditions" that will direct both mouse (D3 and Bruce4) and human (H9) ES cells to differentiate to the airway epithelial cell lineages based on differentiation marker expression that are specific for airway epithelial cells, cell growth, and cell morphology using minus/plus one factor approach at various culture time points. 2) Assess morphologically and immunohistochemically the potential of the airway epithelial cell lineages derived from mouse (D3 and Bruce4) and human (H9) ES cells under the "minimally optimized serum-free culture conditions" to regenerate airway epithelium through the repopulation of denuded rat tracheal graft implanted in SCID/nude mice. 3) Characterize the patterns of localization/differentiation of in vivo transplanted cell lineages derived from GFP-tagged Bruce4 ES cells under the "minimally optimized serum-free culture conditions" during the repair process of the airway epithelium in C57BL/6 mice that have been previously treated with an airway epithelium-specific toxicant, naphthalene. [unreadable] [unreadable] [unreadable]